The gene regulatory network guiding specification of the neurogenic animal plate domain (APD)(55%).[unreadable] Using bioinformatics as well as microarray-based and in situ hybridization screens, we have identified most of the regulatory proteins (50) expressed selectively in the animal plate. We will determine positions of genes with the network by monitoring effects of loss of individual gene functions on quantitative levels and spatial patterns of expression of the others. One of the most interesting genes is FoxQ2, the earliest known APD-specific gene, which we showed this year to be necessary for differentiation of the three known cell types in the APD. Unexpectedly, FoxQ2 also inhibits expression of nodal, which encodes the key signaling molecule that initiates patterning along the secondary axis of the embryo. FoxQ2 is eliminated from the nodal-responsive animal hemisphere by signals dependent on canonical Wnt signaling from underlying vegetal blastomeres. We propose that FoxQ2 provides a checkpoint linking Wnt and nodal signals, which coordinates development long the primary and secondary axes of the embryo. This is a new mechanism that is critical for establishing the major tissue types of the embryo and a manuscript has been submitted for publicatin (Yaguchi, S., Yaguchi, J., Angerer, R.C. and Angerer, L.M. (2007) A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos. Dev. Cell, in review.)[unreadable] [unreadable] The mechanisms of early endoderm specification.(25%) [unreadable] We have discovered that ActivinB signaling through the Alk4/5/7 receptor is the classic signal sent during cleavage stages from micromeres and their progeny, which is required for endoderm development and timely gastrulation. This early signal is thought to be the first nuclear b-catenin-dependent step in sea urchin embryo endoderm development. Loss of either ActivinB or Alk4/5/7 function mimics loss of micromeres and ectopic ActivinB signaling can be induced by ectopic micromeres. We have identified a specific subset of genes in the endoderm gene regulatory network that respond to ActivinB signaling. Because these results alter the current status of the sea urchin endomesoderm gene regulatory network, a major goal is to determine how ActivinB signaling fits into the mechanism underlying endoderm specification. A manuscript is in preparatino (Sethi, A., Angerer, R.C. and Angerer, L.M. (2007) ActivinB/Alk4/5/7 signaling is an early micromere-dependent signal required for early endoderm specification in sea urchin embryos).[unreadable] [unreadable] Bioinformatics.(20%) [unreadable] We continued to improve publicly available computational methods for sequence analysis of sea urchin genes by linking all of the sea urchin gene predictions to GO categories, to temporal expression and transcriptome data. These methods are essential for building the comprehensive networks of genes that control development of different cell types.